Abstract Non-invasive neuroimaging has become a dominant tool in studies of human brain function in health and disease. Currently, PET represents our only tool for non-invasively probing neurotransmitter release, with the majority of such studies focusing on the dopamine system. Despite the exquisite molecular specificity to some of the neurochemical processes underlying brain activation, questions remain about the source of the PET signal and the accuracy of inferences based upon displacement of radiotracer. In order to address these issues, we propose mechanistic studies and better characterization of an agonist radiotracer ([11C]PHNO) that promises better sensitivity to dopamine release. To help interpret the source of signal, our recent work has focused upon combining PET with concurrent fMRI in order to supplement the neurochemical signature provided by PET measurements of receptor occupancy with an fMRI readout describing the functional consequences of that occupancy. In order to set the stage for extracting subtle changes in PET occupancy, we have described a refined PET tracer-kinetic model that should reduce systematic bias. In order to understand the relationship of fMRI signals to changes in occupancy, we have developed simple single and multi-receptor models of dopamine-induced fMRI signal. In accordance with prior PET work that indirectly suggested divergent responses to receptor agonists and antagonists that might be indicative of neuroreceptor trafficking, we have identified different PET/fMRI relationships for agonists versus antagonists. In the proposed studies, we will utilize a mouse knock-out model in conjunction with dopamine microdialysis to more directly test hypotheses about how receptor trafficking influences PET and fMRI signals, and we will perform studies in non-human primates (NHP) on clinical scanners to demonstrate effects of acute and chronic dopamine stimulation using two different radiotracers ([11C]PHNO and [11C]raclopride). We will utilize unilateral deep brain stimulation (DBS) in NHP to produce a unilateral, focal, and titratable model of dopamine release that can be validated by simultaneous fMRI and used to compare the sensitivity of each radiotracer to dopamine release. As a translational complement, we will perform studies in healthy human volunteers to test the magnitude of behaviorally-modulated dopamine using 11C]PHNO and [11C]raclopride and characterize the spatial response versus simultaneously acquired fMRI. The proposed studies will help improve our understanding of PET measurements of endogenous neurotransmitter release and may lead to more robust measurements of behaviorally modulated dopamine release in human subjects.